The reduction of dead/dying neurons represents a critical mechanism for the anti-acute ischemic stroke (AIS) effect of Panax notoginseng, however, its molecular basis remains unclear. Recent findings implicate chemokine-like factor 1 (CKLF1) as a key contributor to the impaired clearance of dying neurons. Here, we established an integrated high-throughput screening strategy combining biolayer interferometry (BLI), liquid chromatography-tandem mass spectrometry (LC-MS/MS), and NanoBRET technologies to identify CKLF1 inhibitors among Panax notoginseng saponins (PNS). Of note, ginsenoside Rg1 (GRg1) exhibits the highest affinity for CKLF1 and the most potent inhibitory efficacy against the CKLF1-CCR4 interaction, effectively suppressing CKLF1-C27 peptide-induced calcium influx and cytokine production. In experimental AIS models, GRg1 confers neuroprotective properties by mitigating ischemic brain damage and promoting neuronal functional recovery. Mechanistically, GRg1 binds to CKLF1 and modulates the mTORC1/TFEB pathway, enhancing lysosomal function and thereby facilitating the clearance of dead/dying neurons. This study presents an efficient approach for the discovery of natural CKLF1 inhibitors and highlights GRg1 as a promising therapeutic candidate for enhancing the clearance of dead/dying neurons in AIS.

Panax notoginseng saponins  /  Chemokine-like factor 1  /  Target-fishing  /  Ginsenoside Rg1  /  Ischemic stroke  /  Neuroinflammation  /  Dead/dying neuron clearance  /  Lysosomal dysfunction